The present invention is related to products formed from Keratin derived from hair. More specifically, the present invention is related to films, sheets, and bulk materials formed from keratin. The present invention is directed to a cross-linked keratin based bulk, film or sheet material for use in biomedical implant, wound dressing, and tissue engineering applications. More specifically, one aspect of the present invention relates to a material based primarily on alpha keratin produced by cross-linking keratin derived from a soluble fraction of keratinous material such as hair.
Chronic wounds can be caused by a variety of events, including surgery, prolonged bed rest, and traumatic injuries. Partial thickness wounds can include second degree burns, abrasions, and skin graft donor sites. Healing of these wounds can be problematic, especially in cases of diabetes mellitus or chronic immune disorders. Full thickness wounds have no skin remaining, and can be the result of trauma, diabetes (e.g., leg ulcers), and venous stasis disease, which can cause full thickness ulcers of the lower extremities. Full thickness wounds tend to heal very slowly. Proper wound care technique, including the use of wound dressings, is extremely important to successful chronic wound management. Chronic wounds affect an estimated four million people a year, resulting in health care costs in the billions of dollars. xe2x80x9cTreatment of Skin Ulcers with Cultivated Epidermal Allografts,xe2x80x9d T. Phillips, O. Kehinde, and H. Green, J. Am. Acad. Dermatol., V. 21, pp. 191-199 (1989).
The wound healing process involves a complex series of biological interactions at the cellular level, which can be grouped into three phases: hemostasis and inflammation, granulation tissue formation and reepithelialization; and remodeling. xe2x80x9cCutaneous Tissue Repair: Basic Biological Considerations,xe2x80x9d R. A. F. Clark, J. Am. Acad. Dermatol., Vol. 13, pp. 701-725 (1985). Keratinocytes (epidermal cells that manufacture and contain keratin) migrate from wound edges to cover the wound. Growth factors such as transforming growth factor-xcex2 (TGF-xcex2) play a critical role in stimulating the migration process. The migration occurs optimally under the cover of a moist layer. Keratins have been found to be necessary for reepithelialization. Specifically, keratin types K5 and K14 have been found in the lower, generating, epidermal cells, and types K1 and K10 have been found in the upper, differentiated cells. Wound Healing: Biochemical and Clinical Aspects, I. K. Cohen, R. F. Diegleman, and W. J. Lindblad, eds., W. W. Saunders Company, 1992. Keratin types K6 and K10 are believed to be present in healing wounds, but not if in normal skin. Keratins are major structural proteins of all epithelial cell types and appear to play a major role in wound healing.
An optimum wound dressing would protect the injured tissue, maintain a moist environment, be water permeable, maintain microbial control, deliver healing agents to the wound site, be easy to apply, not require frequent changes, and be non-toxic and non-antigenic. Although not ideal for chronic wounds, several wound dressings are currently on the market, including occlusive dressings, non-adherent dressings, absorbent dressings, and dressings in the form of sheets, foams, powders, and gels. Wound Management and Dressing, S. Thomas, The Pharmaceutical Press, London, 1990.
Attempts have been made to provide improved dressings that would assist in the wound healing process using biological materials such as growth factors. To date, these biologicals have proven very costly and have shown minimal clinical relevance in accelerating the chronic wound healing process. In cases of severe full thickness wounds, autografts (skin grafts from the patient""s body) are often used. Although the graft is non-antigenic, it must be harvested from a donor site on the patient""s body, creating an additional wound. In addition, availability of autologous tissue may not be adequate. Allografts (skin grafts from donors other than the patient) are also used when donor sites are not an option. Allografts essentially provide a xe2x80x9cwound dressingxe2x80x9d that provides a moist, water-permeable layer, but is rejected by the patient usually within two weeks and does not become part of the new epidermis.
What would be desirable, and has not heretofore been provided, is a wound dressing that protects the injured tissue, maintains a moist environment, is water permeable, is easy to apply, does not require frequent changes, is non-toxic and non-antigenic, and most important, delivers effective healing agents to the wound site.
Film materials compatible with living tissue are useful for a number of applications including tissue engineering scaffolding, diffusion membranes, coatings for implantable devices, and cell encapsulants. Bulk keratin materials compatible with living tissue are useful for a number of applications including open cell tissue engineering scaffolding and bulk, cross-linked biomaterials. Tissue engineering is a rapidly growing field encompassing a number of technologies aimed at replacing or restoring tissue and organ function. The consistent success of a tissue-engineered implant rests on the invention of a biocompatible, mitogenic material that can successfully support cell growth and differentiation and integrate into existing tissue. Such a scaffolding material could greatly advance the state of the tissue engineering technologies and result in a wide array of tissue engineered implants containing cellular components, such as osteoblasts, chondrocytes, keratinocytes, and hepatocytes, to restore or replace bone, cartilage, skin, and liver tissue respectively.
Diffusion membranes are commonly formed of synthetic polymeric materials, rather than biologically-derived materials. Diffusion membranes derived from biological materials have the advantage of enhanced biocompatibility. In particular, non-antigenic diffusion membranes are compatible with implantation in the human body and would provide great advantages in controlled drug release applications.
Implantable devices, such as pacemakers, stents, orthopedic implants, urological implants, dental implants, breast implants, and implants for maxillofacial reconstruction are currently encased in, or made of, materials including titanium, silicone, stainless steel, hydroxyapatite, and polyethylene, or encapsulated in materials such as silicone or polyurethane. These metals, ceramics, and synthetic polymers have disadvantages related to biocompatibility and antigenicity which can lead to problems related to the long term use of these devices. A coating material derived from biological materials and having non-antigenic and mitogenic properties would provide a device the advantage of long term biocompatibility in vivo and potentially extend the useful lifetime of an implant while decreasing the risk of an allergic or negative immune response from the host.
Cell encapsulants such as Chitin/Alginate and bovine-derived collagen are used to encapsulate mammalian cells for applications such as tissue engineering/organ regeneration and bacteria for cloning applications. A non-antigenic, non bioresorbable cell encapsulant material would have the advantages of providing the cell with a mitogen and increasing the chances for the cell to accomplish its tissue engineering function.
A bulk, cross-linked implantable biomaterial that was non-antigenic and possessed the appropriate mechanical properties could be used for maxillofacial restoration, for example, for both soft and hard tissue replacement. Such a bulk material could also be used for orthopedic applications as a bone filler and for cartilage regeneration. A bulk material capable of being implanted could also be used for neurological applications, such as for nerve regeneration guides.
Keratin, often derived from vertebrate hair, has been processed into various forms. Commonly assigned U.S. Pat. No. 5,358,935 discloses mechanically processing human hair into a keratinous powder. The hair is bleached, rinsed, dried, chopped, homogenized, ultrasonicated, and removed from solvent, leaving a keratin powder. In U.S. Pat. No. 5,047,249, Rothman discusses activating keratin with a reducing agent and applying the activated keratin to a wound. Rothman believes the activated keratin thiol groups will react with thiol groups in the wound tissue and form a disulfide bond, allowing the keratin to adhere to and protect the wound.
Keratin derived materials are believed to be non-antigenic, particularly when derived from a patient""s own keratin. A film formed from keratin based material would be desirable. A keratin film able to be used for tissue-engineering scaffolds, diffusion membranes, implantable device coatings, and cell encapsulants would be very useful. A solid keratin bulk material would also have great utility. In addition, a non-antigenic, mitogenic open cell keratin scaffold would prove highly beneficial for use as a tissue engineered scaffold to support, nourish, and stimulate cell growth preceding and following implantation.
The present invention includes a sheet formed of cross-linked keratin not requiring a synthetic binding agent. The sheet is believed to be bound together by reformed disulfide linkages and hydrogen bonds. A preferred use of the sheet is as a wound healing dressing. Another preferred use is as a tissue engineering cell scaffold for implant applications. The sheet can be formed from a combination of soluble and insoluble protein fractions derived from hair, including alpha and beta keratin fractions. Keratin can be obtained from a number of sources, including human or animal hair and finger or toe nails, with one source being hair of the patient or a donor.
The sheet can be formed by providing an insoluble chemically modified keratin fraction suspended in water and lowering the pH until the keratin protein is partially swelled. Partially swelled is defined as the protein molecule swelling such that the resulting suspension of keratin particles behaves like a colloidal suspension. In one method, concentrated sulfuric acid is added until a pH of less than 1 is reached. Applicants believe the low pH disrupts the hydrogen bonds which have been rendering the keratin fraction insoluble, thereby allowing the protein to partially swell. The partially swelled keratin is then made basic with ammonium hydroxide. This treatment exchanges the non-volatile acid with a volatile base, which is removed upon drying. Alternatively, a volatile acid, such as formic acid, may be employed, eliminating the requirement for further treatment with a volatile base. The resulting slurry can then be cast onto a flat surface or mold of appropriate geometry and surface finish and air dried to produce a cross-linked keratin sheet. Applicants believe the cross-links result from the thiol groups reforming disulfide linkages and from the amine, and carboxylic acid groups forming hydrogen bonds.
The resulting sheet is thus formed of pure keratin. Keratin has been shown to be biocompatible, non-immunogenic, not to inhibit activated T-cells and therefore not interfere with the normal cell mediated immune response, and to be mitogenic for keratinocytes, fibroblasts, and human microvascular endothelial cells. Keratin has also been shown to promote epithelialization in wound healing studies on rats and humans.
Another embodiment of the invention includes partially oxidizing the keratin disulfide linkages to form hydrophilic groups. One such method includes treating the keratin with peractic acid to form sulfonic acid groups from a substantial portion, but not all of, the disulfide bonds. Most of the sulfonic acid groups remain in the final product as hydrophilic groups, binding water and hydrating the keratin material. A later reduction step cleaves many of the remaining disulfide bonds to form cysteine residues. The partially oxidized and reduced keratin can then be in put in solution, concentrated, and cast onto a flat surface to oxidize and re-form disulfide cross-links. In one method, oxygen in air acts as the oxidizing agent, with the keratin being air dried to form a film on the flat surface. The moist keratin sheet, consisting primarily of keratin derived from beta keratin, has the consistency of moist, thick paper. The sheet dries to a brittle material, which can be rehydrated to a supple, skin-like material. The rehydrated sheet has the look and feel of skin while retaining moisture within the sheet and within the wound. The sheet can be used as a wound-healing dressing or as a cell-growth scaffold. The sheet can be cut and shaped as needed before being applied to the wound. The keratin sheets provide a non-antigenic wound dressing that maintains wound moisture for migrating epithelial cells and provides a scaffold for cell growth for tissue engineered implants. Other applications for this keratin sheet include use as diffusion membranes and as an encapsulant for cells.
The present invention includes methods for forming keratin based thin films, open cell foams, and bulk materials. The thin films are suitable for use as wound dressings, tissue-engineering scaffolds, diffusion membranes, coatings for implantable devices, and cell encapsulants. In one method, cut, washed, rinsed, and dried vertebrate hair is provided. The hair is reduced with a reducing agent, such that some of the disulfide linkages are broken, and a more soluble keratin fraction and a less soluble keratin fraction formed. The more soluble keratin fraction is separated, collected, and deposited onto a surface, thereby forming a layer of the more soluble keratin fraction. The keratin layer is exposed to an oxidizing agent, such as air, oxygen, or H2O2, and preferably dried. The free thiol groups are oxidized by the oxidizing agent, the resulting keratin film being strengthened by the newly formed disulfide bonds. A higher degree of crosslinking, and therefore strength, can be obtained by the addition of crosslinking agents such as glutaraldehyde.
In one method according to the present invention, a keratin solution is provided, the keratin being dissolved in a first solvent such aqueous thioglycolate. The keratin has free thiol groups, produced by methods such as reduction with ammonium thioglycolate. A second solvent such as hexane or Freon is provided, the second solvent preferably being substantially immiscible in the first solvent and the keratin preferably being substantially insoluble in the second solvent. An emulsion of the second solvent in the keratin solution can be formed using a homogenizer. The emulsion is freeze dried, preferably by freezing the emulsion and removing the first and second solvents under vacuum, creating a porous keratin material. The porous keratin material can be warmed to room temperature in the presence of an oxidizing agent, promoting the formation of disulfide cross-links between the keratin. In one method, the oxidizing agent is an oxygen containing gas such as air. In another method, hydrogen peroxide is mixed with the keratin solution prior to homogenizing. Applicants believe the resulting material is an open cell scaffold having substantially spherical voids corresponding to the second solvent in the emulsion and a cross-linked keratin structure corresponding to the keratin solution in the emulsion.
In another method according to the present invention, a keratin solution is provided, the keratin being dissolved in a solvent such as aqueous thioglycolate. The keratin has free thiol groups, produced by methods such as reduction with ammonium thioglycolate. The keratin can be atomized and sprayed onto a very cold surface, sufficiently cold to freeze the keratin solution. In one method, the surface is the surface of a mold. More keratin solution can be atomized and sprayed over the already frozen keratin, thereby building up a thicker open cell layer of frozen keratin. The frozen keratin can be freeze dried by removing at least a substantial portion of the solvent, and preferably all of the solvent, under low pressure at low temperature. The keratin material can be warmed to room temperature in the presence of an oxidizing agent, promoting the formation of disulfide cross-links within the keratin solids formed and between the keratin solids formed. In one method, the oxidizing agent includes gaseous oxygen. In another method, the oxidizing agent includes hydrogen peroxide added to the keratin solution. Applicants believe the resulting structure is an open cell scaffold having substantially spherical keratin structures corresponding to the atomized keratin and having voids therebetween. Applicants believe the substantially spherical keratin structures have disulfide cross-links formed within, and the structures have disulfide cross-links between structures where touching each other.
In one method, according to the invention, hair is cut, washed, dried, and suspended in ammonium hydroxide containing ammonium thioglycolate. The suspension is under a nitrogen atmosphere. The basic ammonium thioglycolate solution serves to solubilize the keratin and reduce the disulfide cross-links. Cysteine thiol groups and cysteine thioglycolate groups are formed from the broken disulfide bonds. The nitrogen atmosphere serves to prevent oxidation and reformation of disulfide bonds. Heating is preferably followed by comminuting the hair particles with a tissue homogenizer followed by further heating under a nitrogen atmosphere. A fine keratin suspension results.
The fine keratin suspension is centrifuged, and the supernatant containing a more soluble keratin fraction is collected and precipitated out with acid. The precipitate is resuspended in ammonium hydroxide. The keratin solution is then cast as a thin film on a surface and allowed to air dry. The air serves to remove water, concentrate the keratin, and oxidize the cysteine thiol groups, forming disulfide bridges and strengthening the film. Further crosslinking can be achieved using chemical means such as glutaraldehyde. The resulting film is tough and insoluble.
In one method, cut, washed, rinsed, and dried vertebrate hair is provided. The hair is reduced with a reducing agent, such that some of the disulfide linkages are broken, and a more soluble keratin fraction and a less soluble keratin fraction formed. The more soluble keratin fraction is separated, collected, and concentrated, and the more soluble keratin fraction is deposited into a mold. The concentrated keratin solution in the mold is exposed to an oxidizing or crosslinking agent and preferably dried. The free thiol groups are oxidized by the oxidizing agent or cross-linked by the crosslinking agent, and the keratin strengthened by newly formed disulfide bonds. In another method, the concentrated keratin solution is either atomized into a cold mold or mixed with a polar solvent, emulsified, and freeze-dried to form an open-cell material. The keratin solution is exposed to an oxidizing or crosslinking agent, which cross-links and strengthens the material. A porous keratin material remains.
In another method according to the present invention, the more soluble keratin solution is further concentrated, for example, by air drying or heating under sub-ambient pressure. The concentrated solution is poured into a mold and allowed to air dry. The air serves to remove water, concentrate the keratin, and oxidize the cysteine-thiol groups, forming disulfide bridges and strengthening the keratin material. The resulting bulk keratin material is tough and insoluble. In another method, a liquid oxidizing agent such as hydrogen peroxide is used. In yet another method, a crosslinking agent such as glutaraldehyde is used.